Immunopotentiative composition

ABSTRACT

Compositions for cancer or infection treatment via immunopotentiation caused by inhibition of immunosuppressive signal induced by PD-1, PD-L1, or PD-L2 and therapies using them, immunopotentiative substrates included as the active ingredient, screening methods of the substrates for cancer or infection treatment, cell lines used for the screening methods, evaluation methods that selects the substrates for cancer treatment, and carcinoma cell transplanted mammals used for the evaluation methods. 
     The compositions of the present invention that inhibits the function of PD-1, PD-L1, or PD-L2 are useful for cancer or infection treatment.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/245,692, filed Apr. 4, 2014, which is a divisional application ofU.S. application Ser. No. 12/959,307 (now U.S. Pat. No. 8,728,474),filed Dec. 2, 2010, which is a divisional application of U.S.application Ser. No. 12/538,698, filed Aug. 10, 2009 (now U.S. Pat. No.8,168,179), which is a divisional application of U.S. application Ser.No. 10/519,925, filed Jan. 3, 2005 (now U.S. Pat. No. 7,595,048), whichis a National Stage Entry of PCT/JP03/08420, filed Jul. 2, 2003, whichclaims priority to Japanese Patent Application Nos. 2002-194491 and2003-029846, filed Jul. 3, 2002 and Feb. 6, 2003, respectively. Theentire disclosures of the above-listed applications are herebyincorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to immunopotentiation characterized byinhibiting immunosuppressive signals induced by PD-1, PD-L1 or PD-L2,compositions for cancer or infection treatment, and therapies that usethem.

More specifically, the present invention relates to the compositions forcancer or infection treatment through an immunopotentiation caused byinhibition of immunosuppressive signals induced by PD-1, PD-L1 or PD-L2,the therapies that use them, screening methods of immunopotentiativesubstances, or substances for cancer treatment or for infectiontreatment that are contained in the composition as an active ingredient,cell lines used for those screening methods, an evaluation that selectsthe substances for cancer treatment, and carcinoma cells transplantedmammals used for the evaluation.

BACKGROUND ART

Immunotherapies can reduce side reactions that can't be avoided easilyin most chemotherapies, and is expected as a therapy with extremely highspecificity. The Immunotherapies can be executed in the purpose torecover patient's QOL by activating the immune reaction that humanoriginally has by an extrinsic method and subrogating a part of the loadby medication.

Immunopotentiation can be executed by methods of activating immunereaction of T lymphocytes. It is said that not only stimulation throughantigen receptors (TCR) but also an additionally stimulative inducementthrough conjugated stimulative molecular groups (for example, CD28)could be necessary for the activation of T cells. However, it isreported that as the molecular groups with homologous structures to theconjugated stimulative molecular groups, CTLA-4 and PD-1 were discoveredrecently and give signals that suppress signals of antigen receptors(TCR). It is thought that a method of activating T cells could be aneffective mean to suppress the function of those conjugated controlmolecules.

PD-1 was cloned as 55 kD of I type membrane protein that belong to animmunoglobulin family (The EMBO Journal (1992), vol. 11, issue 11, p.3887-3895, JP5336973, JP7291996). Human PD-1 cDNA is composed of thebase sequence shown in EMBL/GenBank Acc. No. NM_005018 and mouse PD-1cDNA is composed of the base sequence shown in Acc. No. X67914, andthose expression are observed when thymus cells differentiate fromCD4−CD8− cell into CD4+CD8+ cell (International Immunology (1996), vol.18, issue 5, p. 773-780, Journal of Experimental Medicine (2000), vol.191, issue 5, p. 891-898.). It is reported that PD-1 expression inperiphery is observed in myeloid cells including T cells or Blymphocytes activated by stimulation from antigen receptors, oractivated macrophages (International Immunology (1996). vol. 18, issue5, p. 765-772.).

In an intracellular domain of PD-1, there are ITIM motifs(Immunoreceptor Tyrosine-based Inhibitory Motif) that could been thoughtto be a repression domain to immune reaction. Since PD-1-deficient micedevelop lupus-like autoimmune disease such as glomerular nephritis andarthritis (for C57BL/6 gene background.) (International Immunology(1998), vol. 10, issue 10, p. 1563-1572., Immunity (1999), vol. 11,issue 2, p. 141-151.) and a disease like dilated cardiomyopathy (forBALB/c gene background.)(Science (2001), vol. 291. issue 5502, p.319-332.), it has been suggested that PD-1 could be a control factor ofdevelopment of autoimmune disease, especially the peripheralself-tolerance.

PD-L1 (human PD-L1 cDNA is composed of the base sequence shown byEMBL/GenBank Acc. No. AF233516 and mouse PD-L1 cDNA is composed of thebase sequence shown by NM_021893.) that is a ligand of PD-1 is expressedin so-called antigen-presenting cells such as activated monocytes anddendritic cells (Journal of Experimental Medicine (2000), vol. 19, issue7, p. 1027-1034.). These cells present interaction molecules that inducea variety of immuno-inductive signals to T lymphocytes, and PD-L1 is oneof these molecules that induce the inhibitory signal by PD-1. It hasbeen revealed that PD-L1 ligand stimulation suppressed the activation(cellular proliferation and induction of various cytokine production) ofPD-1 expressing T lymphocytes. PD-L1 expression has been confirmed innot only immunocompetent cells but also a certain kind of tumor celllines (cell lines derived from monocytic leukemia, cell lines derivedfrom mast cells, cell lines derived from hepatic carcinomas, cell linesderived from neuroblasts, and cell lines derived from breast carcinomas)(Nature Immunology (2001), vol. 2, issue 3, p. 261-267.).

Though PD-L2 (human PD-L2 cDNA is composed of the base sequence shown byEMBL/GenBank Acc. No. NM_025239 and mouse PD-L2 cDNA is composed of thebase sequence shown by NM_021896.) had been identified as a secondligand of PD-1, it has been reported that the expression and functionare almost same as PD-L1 (Nature Immunology (2001), vol. 2, issue 3, p.261-267.).

It has been thought that the inhibitory signals from the conjugatedsuppressive molecules represented by PD-1 could control abnormal immunereaction to autoantigen and immunological tolerance in lymphocytegeneration or maturation by a mechanism that appropriately controlspositive signals with antigen receptors (TCR) and conjugated stimulationmolecules. It has been thought that a certain kind of tumour and viruscould use those conjugated suppressive molecules to intercepte theactivation and proliferation of T cells and weaken the host immunityreaction to oneself by a direct or indirect mechanism (Cell (1992), vol.71, issue 7, p. 1093-1102. Science (1993), vol. 259, issue 5093, p.368-370.). It has been thought that those conjugated suppressivemolecules could have caused the impairment of T cells in a part ofdisease thought to originate in impairment of T cells.

DISCLOSURE OF THE INVENTION

A problem of the present invention is to provide compositions toactivate immunity by inhibiting the inhibitory signals of PD-1, PD-L1 orPD-L2 and compositions for cancer or infection treatment through thismechanism.

The present inventors paid attention to PD-1, PD-L1, or PD-L2 as a newtarget in cancer or infection treatment and found that substances thatinhibit the inhibitory signals of PD-1, PD-L1 or PD-L2 inhibit cancerproliferation through the mechanism of the recovery and activation ofimmune function. Further, they found that PD-1 signal, concretely, aninteraction of PD-1 and PD-L1 or PD-1 and PD-L2 took part in theexclusion of infectious virus. According to those facts, they found thesubstances that could inhibit the inhibitory signals of PD-1, PD-L1 orPD-L2 having therapeutic potential for cancer or infection and completedthe present invention.

That is, the present invention relates to

1. an immunopotentiative composition containing an immunosuppressivesignal inhibitor of PD-1, PD-L1 or PD-L2,2. a composition for cancer treatment containing the immunosuppressivesignal inhibitor of PD-1, PD-L1 or PD-L2,3. the composition for cancer treatment of the subsection 2, which is acomposition that suppresses cancer metastasis,4. a composition for infection treatment containing an immunosuppressivesignal inhibitor of PD-1, PD-L1 or PD-L2,5. the composition for cancer treatment of the subsection 2 or 3, whichis characterized by acting through immunopotentiation,6. the composition for infection treatment of the subsection 4, whichare characterized by acting through immunopotentiation,7. the composition of either of the subsection 1 to 6, which theimmunosuppressive signal inhibitor is/are one or more selected from aninteraction inhibitor of PD-1 and PD-L1 or PD-1 and PD-L2, anintracellular signaling inhibitor of PD-1, and an inhibitory substanceof PD-1, PD-L1 or PD-L2 production,8. the composition of the subsection 7, which is/are one or more of theinteraction inhibitor(s) of PD-1 and PD-L1 selected from PD-1 antibody,PD-L1 antibody, soluble PD-1, and soluble PD-L1,9. the composition of the subsection 8, which is PD-1 antibody selectedfrom anti-human PD-1 antibody of which hybridomas identified byInternational Trust Number FERM BP-8392 product, anti-PD-1 antibodyhumanized from non-human antibody, and human type anti-human PD-1antibody,10. the composition of either of the subsection 1 to 6, which theimmunosuppressive signal inhibitor is a lymphocyte of which PD-1expression is inhibited by gene-recombination.11. the composition of the subsection 7, which the interaction inhibitorof PD-1 and PD-L1 or PD-1 and PD-L2, an intracellular signalinginhibitor of PD-1, or the inhibitory substance of PD-1, PD-L1 or PD-L2production is one or more of substance(s) selected from protein,polypeptide or peptide, polynucleotide or polynucleoside, antibody orthe derivative, organic synthesis compound, inorganic compound, andnatural product,12. an immunopotentiative method containing a method of administeringthe immunosuppressive signal inhibitor of PD-1, PD-L1 or PD-L2,13. a method for cancer treatment containing a method of administeringthe immunosuppressive signal inhibitor of PD-1, PD-L1 or PD-L2,14. the method of cancer treatment of the subsection 13, which is amethod of suppressing cancer metastasis,15. a method for infection treatment containing a method ofadministering the immunosuppressive signal inhibitor of PD-1, PD-L1 orPD-L2,16. the method for cancer treatment of the subsection 13 or 14, which ischaracterized by acting through immunopotentiation,17. the method for infection treatment of the subsection 15, which ischaracterized by acting through immunopotentiation,18. the method of either of the subsection 12 to 17, which theimmunosuppressive signal inhibitor is/are one or more selected from theinteraction inhibitor of PD-1 and PD-L1 or PD-1 and PD-L2, theintracellular signaling inhibitor of PD-1, and the inhibitory substanceof PD-1, PD-L1 or PD-L2 production,19. the method of the subsection 18, which the interaction inhibitoris/are one or more selected from PD-1 antibody, PD-L1 antibody, solublePD-1, and soluble PD-L1,20. the method of the subsection 19, which PD-1 antibody is an antibodyselected from anti-human PD-1 antibody of which hybridomas identified byinternational trust number FERM BP-8392 product, anti-PD-1 antibodyhumanized from non-human antibody, and human type anti-human PD-1antibody,21. the method of either of the subsection 12 to 17, which theimmunosuppressive signal inhibitor is a lymphocyte of which PD-1expression is inhibited by gene-recombination.22. the method of the subsection 18, which the interaction inhibitor ofPD-1 and PD-L1 or PD-1 and PD-L2, the intracellular signaling inhibitorof PD-1, or the inhibitory substance of PD-1, PD-L1 or PD-L2 productionare one or more of substance(s) selected from protein, polypeptide orpeptide, polynucleotide or polynucleoside, antibody or the derivative,organic synthesis compound, inorganic compound, and natural product,23. use of the immunosuppressive signal inhibitor of PD-1, PD-L1 orPD-L2 to manufacture the immunopotentiative composition,24. use of the immunosuppressive signal inhibitor of PD-1, PD-L1 orPD-L2 to manufacture the composition for cancer treatment,25. use of the substance of the subsection 24, which the composition forcancer treatment is the composition for suppression of cancermetastasis,26. use of the immunosuppressive signal inhibitor of PD-1, PD-L1 orPD-L2 to manufacture the composition for infection treatment,27. carcinoma cell lines for screening, which are transformed to expressPD-L1 or PD-L2,28. a screening method for the immunopotentiative substancecharacterized by touching the cells of the subsection 27 to lymphocytesand a subject substance, followed by evaluating the enhancement of thesubject substance for immune reaction of lymphocytes to the cells of thesubsection 27,29. a screening method for a substance for cancer treatmentcharacterized by touching the carcinoma cells of the subsection 27 tolymphocytes and a subject substance, followed by evaluating theenhancement of the subject substance for immune reaction of lymphocytesto carcinoma cells and inhibitory effect on the subject substance forcarcinoma cells proliferation,30. a screening method for a substance for infection treatmentcharacterized by touching the infected cells of the subsection 27 tolymphocytes and a subject substance, followed by evaluating theenhancement of the subject substance for immune reaction of lymphocytesto the infected cells and inhibitory effect on the subject substancesfor pathogens proliferation,31. a mammal created by transplant the carcinoma cell lines of thesubsection 27, and32. a screening method for a substance for cancer treatmentcharacterized by administering the subject substance to the mammal ofthe subsection 31, followed by evaluating the inhibitory ratio of thesubject substance for the transplanted carcinoma cells proliferation.

PD-1, PD-L1, or PD-L2 of the present invention includes each one derivedfrom mammal, for example, mouse, rat, hamster, guinea pig, dog, pig,ape, or primate including human. They are suitable to be human PD-L1,PD-1, and PD-L2, respectively.

The immunosuppressive signal of PD-1, PD-L1, or PD-L2 in the presentinvention is at least composed of the interaction of PD-1 and PD-L1 orPD-1 and PD-L2, and the intracellular signalings of PD-1. Production ofPD-1, PD-L1 or PD-L2 itself is included in them.

The immunosuppressive signal of PD-1, PD-L1, or PD-L2 in the presentinvention is inhibited by direct or indirect inhibition of theinteraction of PD-L1 or PD-1 and PD-L2 or the intracellular signalingsof PD-1. A substance that selectively binds to PD-1, PD-L1, or PD-L2respectively is included as a substance with those inhibitoryactivities. For example, it is suitable to be protein, polypeptide orpeptide, polynucleotide or polynucleoside, antibody or the derivative,organic synthesis compound, inorganic compound, or natural product.Especially, an antibody to PD-1, PD-L1 or PD-L2 is enumerated as anexcellent substance in specificity.

The immunosuppressive signals are inhibited by inhibiting production ofPD-1, PD-L1 or PD-L2 itself.

As an antibody to PD-1, PD-L1 or PD-L2, all antibodies derived fromhuman, mouse, rat, rabbit, or goat which can inhibit theimmunosuppressive signals by PD-1, PD-L1, or PD-L2, those polyclonal ormonoclonal antibodies, complete or shorten (for example, F(ab′) 2, Fab′,Fab, or Fv fragment) antibodies, chimeric antibodies, humanizedantibodies, or completely humanized antibodies will be acceptable.

Such antibodies can be manufactured using a partial protein of theextracellular region of PD-1, PD-L1, or PD-L2 as an antigen according towell-known production methods of antibody or antiserum. The partialprotein of the extracellular region can be prepared by well-knownprotein expression and purification techniques.

The polyclonal antibodies can be manufactured according to well-knownmethods. For example, they can be manufactured by separation andrefinement of the antibody of which a mixture of an antigen and acarrier protein is immunized to suitable animal, and an antibodyinclusion to the antigen is gathered from the immunized animal. As suchanimal, mouse, rat, sheep, goat, rabbit, and guinea pig are generallyenumerated. To improve the antibody producibility, Freund's completeadjuvant or Freund's incomplete adjuvant can be administered with theantigen. The administering is usually executed once every two weeksabout 3-10 times in total. The polyclonal antibody can be gathered fromthe immunized animal's blood and peritoneal fluid, etc. by the abovemethod. The measurement of the polyclonal antibody's titer in antiserumcan be measured by ELISA. The separation and refinement of thepolyclonal antibody can be executed by refining techniques that useactive adsorbents such as antigen binding solid phase, protein A, orprotein G, etc., salting-out, alcohol precipitation, isoelectricprecipitation, electrophoresis, adsorption and desorption with ionexchanger, ultracentrifugation, or separation and refinement ofimmunoglobulins such as gel filtration technique, etc.

As an antibody preparation, the monocional antibody or the modifier ismore suitable.

The monoclonal antibody producing cells can be prepared as hybridomas tobe possible to subculture which produce the monoclonal antibody byselecting the individual of which the antibody titre is confirmed in anantigen immunized animals, gathering the spleen or the lymph node on day2-5 after the final immunization, and fusing the antibody productingcells included in them with homogeneous or heterozoic myeloma cells. Theantigen itself or with the carrier and the diluent is administered tothe part in which the antibody production is possible. To improve theantibody producibility, Freund's complete adjuvant or Freund'sincomplete adjuvant can be administered with the antigen. According tothe method of calling “DNA immunization”, animals are immunized. Thismethod is a method using a phenomenon in which antigen-expressingvectors are introduced into the part and are taken into myocytes on theprocess of tissue repair, and expresses the antigenic protein (NatureImmunology (2001), vol. 2, issue 3, p. 261-267) after Cardiotoxin istreated to immune animal's tibialis anterior muscle of hind leg.

As an immune animal, mouse, rat, sheep, goat, rabbit, or guinea pig canbe used, mouse and rat are suitable. The fusion operation can beexecuted by the method (Nature (1975), vol. 256, issue 5517, p.495-497.) of Kohler and Milstein, and as fusion accelerants,polyethylene glycol (PEG) and Sendai virus, etc. are used. As thosemyeloma cells, myeloma cells such as P3U1, NS1, SP2/0, and AP1 can beused, P3U1 are often used usually. The monoclonal antibody producingcells can be selected by detecting by ELISA, etc. executed by addinghybridoma culture supernatant to solid phase in which antigenic proteinsare adsorbed direct or with carrier perhaps. Hybridoma culturesupernatant's antibody titre can be measured by ELISA. The separationand refinement of the monoclonal antibody can be executed according tothe separation refining method similar to the separation and refinementof immunoglobulin for the above polyclonal antibody. Concretely, it isanti-mouse PD-L1 antibody producted by hybridomas identified byInternational Trust Number FERM BP-8396 or anti-human PD-1 antibodyproducted by a hybridoma identified by International Trust Number FERMBP-8392.

The hybridomas identified by International Trust Number FERM BP-8392 hadbeen deposited as Trust Number FERM P-19162 to National Institute ofAdvanced Industrial Science and Technology, International PatentOrganism Depositary in Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan(ZIP code 305-8566) at Dec. 19, 2002, and had been transferred toInternational Deposit at Jun. 5, 2003. The hybridomas identified byInternational Trust Number FERM BP-8396 had been deposited as TrustNumber FERM P-18908 at Jun. 25, 2002, and had been transferred toInternational Deposit at Jun. 11, 2003.

The antibody fragment means F(ab′)₂. Fab′, Fab, or scFv antibodyfragment and can be prepared by reducing optionally after processingwith protease enzyme.

F(ab′)₂ antibody fragments can be purified by either method of affinitychromatography such as ion-exchange chromatography, gel filtration, orprotein A or protein G column, etc. after the purified monoclonalantibody is completely digested by pepsin. Because digestive time bypepsin is different depending on Ig subtype, it is necessary to prepareit suitably. Fab′ antibody fragment can be prepared by reducing F(ab′)₂by 2-mercaptoethylamine partly. Fab antibody fragment can be prepared bythe direct digestion under the presence of cysteine by the digestiveenzyme papain followed by refining.

Further, the monoclonal antibody is prepared as a rearrangement antibodyand a hybrid antibody modified by gene recombination technology usingDNA sequence that codes the amino acid sequence of antibody isolatedfrom the hybridomas. For example, it can be prepared as a single-chainantibody but not a usual complete type antibody. scFv antibody (singlechain Fv) can be prepared by the method of Jost (Journal of BiologicalChemistry (1994), vol. 269, issue 42, p. 26267-26273.). The single-chainantibody with the characteristic and the affinity of an originalantibody can be prepared by making expression an expression vectorincluding a fused DNA of which DNA fragments respectively coding avariable region of the heavy chain and the light chain had beenconnected with a spacer coding neurtal amino acids (glycine or serine)in suitable host cells.

When non-human antibody is used to treat for human, it is indispensableto decrease the antigenicity of the antibody. Since the immune reactionto patient's antibody often shortens an effective treatment period, theprocess of decreasing the antigenicity of the antibody by making theantibody humanize or completely human type is necessary. The humanizedantibody modified to be acceptable for administering to human is theantibody which is modified so that the decrease of antigenicity or theblood movement may improve to extent that can be allowed in pharmacologywhen the antibody is administered to human.

Human PD-1 antibody or human PD-L1 antibody in specification of thepresent invention includes the humanized or the complete human typeantibody, too.

The humanized antibody can be prepared by substituting a part ofnon-human antibody which was prepared by being immunized to mammalsother than human for a part of human antibody. Concretely, it has beenknown to be able to be prepared by constructing a chimera with a genecoding a constant region of the human antibody (Proc. Natl. Acad. Sci.(USA) (1987), vol. 84, p. 3439-3443, Journal of Immunology (1987), vol.139, issue 1, p. 3521.). The DNA sequence of the human constant regionhas been described to a prior art and the constant region gene can beeasily obtained from an already-known clone. Then, the DNA sequencecoding the variable region of the antibody can be fused to the humanconstant region. An isotype of the human constant region can be selecteddue to desire effective function or antibody dependent cytotoxicity. Asuitable isotype is IgG1, IgG3, and IgG4. Both human light-chainconstant region, K chain, and A chain can be used. It is possible tomake this humanized chimeric antibody expression by a routine method.

The complete human type antibody can be prepared by using mice(XenoMouse (Chemical Biology (2000), vol. 7, issue 8, p. R185-6.),HuMAb-Mouse (Infection and Immunity (2002), vol. 70, issue 2, p.612-9.), TC mouse (Biotechnology and Genetics Enginnering Review (2002),vol. 19, p. 73-82.), and KM mouse (Cloning Stem Cells (2002), vol. 4,issue 1, p. 91-102.)) of which a constant region gene of humanimmunoglobulin have been transferred, and a target antibody can bemass-produced by making the antibody production lymphocytes separatedfrom mice to hybridomas. It can be prepared by phage display method(FEBS Letter (1998), vol. 441, p. 20-24.). In this method, by usingphages of which the human antibody gene have been incorporated into acyclic single strand DNA, the human type antibody can be expressed onthe surface of the phage as a form fused with coat protein of thephages.

Polypeptides or the derivatives that bind to PD-1, PD-L1, or PD-L2include each partial proteins of PD-1, PD-L1 or PD-L2 of which theimmunosuppressive signal is not induced. The presence of PD-1 in theneighborhood of the antigen receptors is indispensable for theinducement of the immunosuppressive signal of PD-1, for that purpose itis necessary to be restrained by the interaction with PD-L1 or PD-L2 inantigen-presenting cells, tumours. or carcinoma cells. Therefore,soluble PD-L1 or soluble PD-L2 with a part which is only extracellulardomains and interacts with PD-1 can inhibit the immunosuppressive signalof PD-1. On the other hand, soluble PD-1 with a part which has a similarstructure and can interact with PD-L1 or PD-L2 can inhibit theimmunosuppressive signal. These soluble proteins have only to include anextracellular region which is necessary and sufficient to bind to PD-1,PD-L1, or PD-L2 and can be prepared by a well-known expression andrefining technique.

If an interaction inhibitor of PD-1 and PD-L1 or PD-1 and PD-L2 is aprotein or a polypeptide and an essential area for the interaction iscomposed by only consecutive polypeptide, such a polypeptide fragmentcan become a mutual antagonist. Further, an antagonist with strongeractivity can be identified from molecular groups of which thispolypeptide fragment is chemically modified, or designed by computerbased on the spatial structure of the polypeptide fragment. Also, thebest antagonist can be more efficiently selected from molecular groupsdesigned by computer based on protein stereoanalysis data of theinteraction area.

A substance that inhibits the interaction of PD-1 and PD-L1, or PD-1 andPD-L2 can be screened directly. Such a substance can be identified fromeach libraries of protein, polypeptide or peptide, polynucleotide orpolynucleoside, non-peptide compound, organic synthesis compound, ornatural product (for example, fermentational product, cell extract,plant extract, and animal tissue extract.).

Since the inhibitory signals of PD-1 intracellular domain are caused bycontacting dephosphorylation enzymes (for example, SHP-1 and 2 (SathishJ G, Journal of Immunology (2001), vol. 166, issue 3, p. 1763-70.)) thatbind to ITIM in PD-1 intracellular domain with an intracellular complexincluding an antigen receptor complex, they are generally inhibited byinhibiting the contact between the antigen receptor complex and PD-1.The substance that inhibits the inhibitory signals includes a substancethat directly inhibits the activity of dephosphorylation enzymes, asubstance that inhibit phosphorylation of tyrosine residue in ITIM, asubstance that inhibits bonds of dephosphorylation enzymes to ITIM, or asubstance that directly inhibits activity of dephosphorylation enzymes,etc. The antigen receptor complex includes T cell receptor complex or Bcell receptor complex.

The production of PD-1, PD-L1, or PD-L2 is inhibited by specificpolynucleotide or polynucleoside, organic synthesis compound, inorganiccompound, or natural product, etc. Especially, the suitablepolynucleotide or polynucleoside includes antisense nucleotidederivative so-called ribozyme. This uses a mechanism of which theexpressed mRNA is destroyed by which the polynucleotide derivative thatis complement to mRNA of PD-1, PD-L1, or PD-L2 is transferred intosubject cells. Further, the vector can be used for the gene manipulationto lymphocyte stem cells gathered from a patient so that it inhibits theexpression of PD-1, and the manipulated cells can be used for cellmedical treatment of which the cells will be proliferated,differentiated, activated, and administered to the patient again.Especially, in immunotherapy for cancer, more specific and clonallymphocytes to target cells can be prepared by adding a specific antigenof the target cells on the process of the maturation and activation oflymphocyte stem cells.

The screening method of the present invention can be executed by amethod of measuring cell function. The carcinoma cell lines forscreening of which PD-L1 or PD-L2 used by the method is transformed tobe expressed includes carcinoma cell lines transformed transitory orstably after expression vectors constructed to express PD-L1 or PD-L2have been introduced to the cells by well-known methods. As thecarcinoma cell lines used, ape COS-1 cells, COS-7 cells, Vero, chinesehamster CHO cells (hereafter, abbreviated with CHO cells.), dhfr genedeficient chinese hamster CHO cells (hereafter, abbreviated with CHO(dhfr-) cells.), mouse L cells, mouse AtT-20, mouse myeloma cells, ratGH3, HEK293 T cells, and human FL cells, etc. are used. Especially, thetransformation of animal cells can be executed according to methodsdescribed in, for example, Cell Technology separate volume 8, NewExperimental Protocol of Cell Technology (1995), vol. 263, published bySHUJUNSHA Co., Ltd., and Virology (1973), vol. 52, issue 456.

Cells naturally expressing PD-L1 or PD-L2 can be also used. Such cellsinclude leukocyte, suitablely, monocytes, macrophages orantigen-presenting cells, epithelial cells, tumor cells, carcinomacells, or those cell lines, etc. As tumor cells or carcinoma cells, forexample, P38D1 cells, P815 cells, NB41A3 cells, MDA-231 cells, SKBR-3cells, MCF-7 cells, BT474 cells, J558L cells, P3U1 cells, PAI cells, X63cells, or SP2/0 cells can be used. Cells infected with pathogens, whichexpress PD-L1 or PD-L2 naturally or compellingly, can be used.Infectious pathogens include human hepatitis virus (hepatitis B,hepatitis C, and hepatitis A) or hepatitis E), human retrovirus, humanimmunodeficiency virus (HIV1 and HIV2), human T cell leukemia virus,human T lymphocytic virus (HTLV1 and HTLV2), simple herpes virus type 1or 2, Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, humanherpesvirus including human herpesvirus 6, poliovirus, measles virus,rubella virus, Japanese encephalitis virus, mumps virus, influenzavirus, adenovirus, enterovirus, rhinovirus, virus developing severelyacute respiratory syndrome (SARS), Ebola virus, West Nile virus, orthese virus modified artificially. Other pathogens include, for example,pathogenesis protozoan (for example, trypanosome, malaria, andtoxoplasma), bacillus (for example, mycobacterium, salmonella, andlisteria) or fungus (for example, candida), etc.

The lymphocytes used in the screening method of the present inventionare T or B lymphocytes, and suitably cytotoxic T lymphocytes (CTL). Theimmune reaction of lymphocytes in the screening method of the presentinvention includes citotoxic response (for example, tumor immunityreaction), mixed lymphocyte reaction, production of cytokines,antibodies, complements or other cell surface antigens, or cellularproliferation, etc.

In the present invention, the screening method of an active ingredientcontained in the composition for immunopotentiation or cancer treatmentcan be executed by measuring cytotoxic activity of cytotoxic Tlymphocytes against subject cells followed by measuring an effect of asubject substance against the activity. This method is an assay ofrecovery and reinforcement of cytotoxic activity by adding the subjectsubstance to a culture mixed cytotoxic T lymphocytes (CTL) naturallyexpressing PD-1 or cell lines (for example, 2C cells) with cellsnaturally or compulsorily expressing PD-L1 or PD-L2 which are derivedfrom syngeneic mouse. Since the cytotoxic activity against PD-L1 orPD-L2 expressing cells is lower than that against PD-L1 or PD-L2non-expressing cells, this method has a feature of which the recovery ofcytotoxic activity (rising part) due to the subject substance can bemeasured more clearly. The recovery of cytotoxicity due to the subjectsubstance can be evaluated as an equivalent to inhibition of suppressionof cytotoxicity. Further, it is preferable that the the cytotoxicity dueto the subject substance is arbitrarily measured. As these cells, tumorcell lines or carcinoma cell lines naturally expressing PD-L1 or PD-L2(Nature Immunology (2001), vol. 2, issue 3, p. 261-267.), for example,P38D1 cells, P815 cells, NB41A3 cells, MDA-231 cells, SKBR-3 cells,MCF-7 cells, BT474 cells, J558 L cells, P3U1 cells, PAI cells, X63cells, or SP2/0 cells can be used, and tumor cell lines or carcinomacell lines transformed so as to stably or transiently express PD-L1 orPD-L2 can be also used.

On the other hand, it is preferable that the cytotoxic lymphocytes arePD-1 expressing cells derived from syngeneic animal to targeted cells.

In the present invention, the screening method of an active ingredientcontained in the composition for infection treatment is an assay ofenhancement effect on immune reaction of lymphocytes to infected cellsor inhibitory effect on proliferation activity of pathogen or virus byadding the subject substance to a culture mixed cytotoxic T lymphocytes(CTL) or cell lines (for example, 2C cells) naturally expressing PD-1with cells naturally or compulsorily expressing PD-L1 or PD-L2 which arederived from syngeneic mouse and was infected with pathogen or virus.

Further, in evaluations using a similar principle, a mammal created bytransplanting the above carcinoma cell lines for screening which aretransformed so as to express PD-L1 or PD-L2 or cells naturallyexpressing PD-L1 or PD-L2 to a syngeneic mammal can be used. To amanufacture method of the mammal, a process of transplanting cells and aprocess of breeding the mammal until becoming appropriate for theevaluation is indispensable. This evaluation is characterized inevaluating proliferation of the transplant cells and the amount ofproduction of various cytokines or cell surface antigens, especially, incase of carcinoma cells, proliferation, permeation, or histologicalanalysis of metastasis of the cells, or survival rates of thetransplanted mammal. The cellular proliferation can be evaluated as thenumber of carcinoma cells per unit capacity in case of ascites tumors orblood cancers, or the size or the weight after removing in case of solidcancer. The effect of the subject substance for cancer treatment in thismethod can be evaluated as an equivalent to effect due to inhibition ofsuppression of cytotoxicity caused in PD-L1 or PD-L2. As such cells,syngeneic cells to a mammal for transplant, with good proliferation aremore suitable. The mammal include s primates except human, mouse, rat,hamster, guinea pig, dog, pig, and ape.

INDUSTRIAL AVAILABILITY

As a substance that demonstrates remarkable suppression of cancerproliferation and life prolongation of an individual by administering tothe carcinoma cells-transplanted animal model, the present inventorsinvented each specific antibody (anti-PD-1 antibody and anti-PD-L1antibody) that inhibited PD-1 and PD-L1 function respectively. Theseantibodies showed actions that recover or reinforce cytotoxic activitythat has relatively decreased by presenting PD-L1 ligand toPD-1-expressing CTL (cytotoxic T lymphocyte) (example 1 and FIG. 1.).This suggests that the cytotoxic activity to carcinoma cells by CTLcould be reinforced by administering these antibodies. In the carcinomacells-transplanted animal model (Protein, Nucleic acid, and Enzyme(1981), vol. 26, issue 3, p. 208-22.) which uses syngeneic mouse ofwhich cell lines artificially expressing PD-L1 derived from mastocytomashave been imported, administration of anti-PD-L1 antibody presentedsuppression of proliferation, invasion, and metastasis of carcinomacells, and life prolongation of an individual (FIGS. 2 and 3.). It wassuggested that inhibition of PD-1 function or production could achievean effect similar to the effect on inhibition of PD-L1 function by thisantibody. This is based on non-proliferation of imported carcinoma cellsin the cancer imported model using PD-1-deficient mice, and presentsthat inhibition of PD-1 function or production could be effective oncancer treatment (Example 5 and FIG. 3.).

Actually, it had been proven that administration of anti-PD-1 antibodysignificantly suppressed metastasis of imported carcinoma cells to liverin the cancer imported animal model (Example 13).

The inventors presented that the substance that inhibits theimmunosuppressive signal induced by PD-1, PD-L1 or PD-L2 was useful forinfection treatment (Example 11, FIGS. 15 and 16).

These results experimentally presented by the present inventors provethat only PD-1 antibody or PD-L1 antibody don't presents aforementionedeffect, but also any substance that can inhibit the immunosuppressivesignal from PD-1, PD-L1, or PD-L2 present almost similar effect. Thesubstances with such effects include, for example, anti-PD-L2 antibody,soluble PD-1, soluble PD-L1, soluble PD-L2, PD-1 antagonists, PD-L1antagonists, PD-L2 antagonists, substances that inhibits interactionbetween PD-1 and PD-L1 or PD-1 and PD-L2, PD-1 production inhibitors,PD-L1 production inhibitors, PD-L2 production inhibitors, andintracellular inhibitory signal inhibitors by PD-1.

Cancer or tumour of which the therapeutic potential could be expected byadministration of the composition for cancer treatment of the presentinvention include, for example, carcinoma, squamous carcinoma (forexample, cervical canal, eyelid, tunica conjunctiva, vagina, lung, oralcavity, skin, urinary bladder, tongue, larynx, and gullet), andadenocarcinoma (for example, prostate, small intestine, endometrium,cervical canal, large intestine, lung, pancreas, gullet, intestinumrectum, uterus, stomach, mammary gland, and ovary). Further, theyinclude sarcomata (for example, myogenic sarcoma), leukosis, neuroma,melanoma, and lymphoma.

The effect on the ones that remarkably express PD-L1 or PD-L2 in thesecancers or tumours is remarkable. PD-L1 or PD-L2 expression can beidentified by checking up surgically excised cancer, tumour mass, orlesioned part gathered outside of the body as a sample. Administrationof the composition of the present invention will become an efficient andavailable method as a treatment after surgery for tumour or cancerpatient that PD-L1 or PD-L2 is remarkably expressed. PD-L1 or PD-L2expression can be identified by, for example, immunochemical methodusing PD-L1 antibody or PD-L2 antibody, RT-PCR, or DNA array.

A side reaction violently decreasing lymphocytes proliferation inchemotherapy and radiotherapy for cancer is inevitable. Administrationof the composition of the present invention presents an effect onstimulating and proliferating the decreased lymphocytes and can suppressa fierce side reaction accompanied by usual chemotherapy to minimum.Further, it is similar as to radiotherapy. Concomitant use with thecomposition of the present invention can greatly decrease the dose ofchemotherapy drug or the exposure dose of irradiation from the dose orthe exposure dose used usually.

The composition for cancer treatment of the present invention can beused together with existing chemotherapy drugs or be made as a mixturewith them. Such a chemotherapy drug include, for example, alkylatingagents, nitrosourea agents, antimetabolites, antitumor antibiotics,alkaloids derived from plant, topoisomerase inhibitors, hormone therapymedicines, hormone antagonists, aromatase inhibitors, P-glycoproteininhibitors, platinum complex derivatives, other immunotherapeutic drugs,and other anticancer agents. Further, they can be used together withhypoleukocytosis (neutrophil) medicines that are cancer treatmentadjuvant, thrombopenia medicines, antiemetic drugs, and cancer painmedicines for patient's QOL recovery or be made as a mixture with them.

The composition for cancer treatment of the present invention can beused together with immunopotentiative substances or be made as a mixturewith them. Such immunopotentiative substances include, for example,various cytokines and a tumor antigen, etc. Cytokines that stimulateimmune reactions include, for example, GM-CSF, M-CSF, G-CSF,interferon-α, β, γ, IL-1, IL-2, IL-3, and IL-12, etc. B7 ligandderivatives, anti-CD3 antibodies and anti-CD28 antibodies, andanti-CTLA-4 antibodies can also improve the immune reactions.

Administration of the tumor antigen can also improve a specific immunereaction against T lymphocytes against carcinoma cells and additionallyor synergistically reinforce by using together with the composition forcancer treatment of the present invention. The tumor antigen can beprepared as a purified protein in case of an already-known gene, or as alysate of carcinoma cells in case of an unknown gene.

Such the tumor antigen includes, for example, HLA-A1 and HLA-A2restrictive peptides of malignant melanoma MAGE-1 or MAGE-3, MART-1, andgp100. Further, they include HER2/neu peptide of mammary carcinomas andovarian carcinomas, MUC-1 peptide of adenocarcinoma, and NY-ESO-1 ofmetastatic carcinomas.

It has been thought that viruses could use conjugate suppressive factorsof T lymphocytes as one of methods to escape from host'simmuno-protection. It is thought that a part of virus infection couldattribute the escape function of such viruses and administration of thecomposition of the present invention could improve the immune reactionof T lymphocytes to viruses.

Administration of the composition for infection treatment of the presentinvention is effective on treatment for, for example, human hepatitisvirus (hepatitis B, Hepatitis C, hepatitis A, or hepatitis E), humanretrovirus, human immunodeficiency virus (HIV1, HIV2), human T leukemiavirus (HTLV1, HTLV2), or human lymphocytic cell type virus. Further, itis thought to be effective on treatment for simple herpes virus type 1or 2, epstein-barr virus, cytomegalovirus, varicella-zoster virus, humanherpesvirus including human herpesvirus 6, poliovirus, measles virus,rubella virus, Japanese encephalitis virus, mumps virus, influenzavirus, adenovirus, enterovirus, rhinovirus, virus developing severelyacute respiratory syndrome (SARS), ebola virus, west nile virus, orthese virus modified artificially.

It is thought to be also effective on infection treatment for otherpathogens such as, for example, pathogenesis protozoan (for example,trypanosome, malaria, and toxoplasma), bacillus (for example,mycobacterium, salmonella, and listeria) or fungus (for example,candida), etc.

The composition for infection treatment of the present invention can beused together with existing anti-HIV drugs, antiviral agents, antibioticagents, antimicrobial agents, or visceral mycosis medicines or be madeas a mixture with them. The anti-HIV drugs include, for example, reversetranscriptase inhibitors (for example, AZT, ddl, 3TC, and d4T), proteaseinhibitors (for example, saquinavir mesylate, ritonavir, nelfinavirmesylate, amprenavir, delavirdine mesylate, saquinavir, andlopinavir/ritonavir) or CCR5 receptor antagonists. The antiviral agentsinclude, for example, anti-herpesvirus agents, anti-influenza virusagents, interferon-α and β, or various immunoglobulins.

The composition for infection treatment of the present invention can beused together with vaccines of virus or pathogen or be made as aformulation with them. Such vaccines include, for example, poliovaccine,measles vaccine. Japanese encephalitis vaccine, BCG vaccine, triplevaccine, mumps virus vaccine, varicella virus vaccine, influenzavaccine, hepatitis A vaccine, hepatitis B vaccine, and cholera vaccine.

The composition of the present invention is usually administeredsystemically or locally, and orally or parenterally.

The dosage is determined depending on medicines used for the presentinvention, age, body weight, symptom, therapeutic effect, administrationroute, and duration of the treatment, etc. For oral administration,generally, the dosage range from 1 μg to 100 mg per an adult is orallyadministered once to several times per day, or the dosage range from 0.1ng to 10 mg per an adult is administered once to several times per dayparenterally, suitably intravenously, and is intravenously administeredfor 1 to 24 hours per day continuously.

Since the dosage changes depending on various conditions as describedabove, there are cases in which doses lower or greater than the abovedosage may be used.

When a concomitant drug of the composition of the present invention andother medicines is administered, it is used as solid medicines forinternal use, and injections, external preparations, suppositoriums,inhalant, pernasal preparation, etc. for parenteral administration.

The solid medicines for oral administration include compressed tablets,pills, capsules, dispersing powders, granules, etc. The capsules includehard capsules and soft capsules. The tablets include sublingual tablets,intraoral strapping tablets, and intraoral speed disintegrators, etc.

As to such the solid medicines, one or more active compound(s) may bepharmaceutically manufactured as itself/themselves or a formulation withexcipients (lactose, mannitol, glucose, microcrystal cellulose, andstarch, etc.), binders (hydroxypropylcellulose, polyvinyl pyrrolidone,and magnesium metasilicate aluminate, etc.), disintegrators (cellulosecalcium glycolate, etc.), lubricants (magnesium stearate etc.),stabilizers, or solubilizers (glutamate and aspartic acid, etc.), etc.according to usual methods. Further, they may be optionally coated bycoatings (sucrose, gelatin, hydroxypropylcellulose, andhydroxypropylmethylcellulose phthalate, etc.) or coated in the layer oftwo or more. Further, capsules of absorbable materials such as gelatinmay be included.

The sublingual tablets are manufactured according to a well-knownmethod. For example, they may be pharmaceutically manufactured as amixture one or more active compound(s) with excipients (lactose,mannitol, glucose, microcrystal cellulose, colloidal silica, and starch,etc.), binders (hydroxypropylcellulose, polyvinylpyrrolidone, andmagnesium metasilicate aluminate, etc.), disintegrator (starch,L-hydroxypropylcellulose, carboxymethylcellulose, crosscarmellosesodium, and cellulose calcium glycolate, etc.), lubricants (magnesiumstearate etc.), swelling agents (hydroxypropylcellulose,hydroxypropylmethylcellulose, carbopole, carboxymethylcellulose,polyvinyl alcohol, xanthan gum, and Cyamoposis Gum, etc.), swellingadjuvants (glucose, fructose, mannitol, xylitol, erythritol, maltose,trehalose, phosphate salt, citrate, silicate, glycine, glutamate, andarginine, etc.), stabilizers, solubilizers (polyethylene glycol,propylene glycol, glutamate, and aspartic acid, etc.), or spices(orange, strawberry, mint, lemon, and vanilla, etc.), etc. according tousual methods. Further, they may be optionally coated by coatings(sucrose, gelatin, hydroxypropylcellulose, andhydroxypropylmethylcellulose phthalate, etc.) or coated in the layer oftwo or more. Additives such as preservatives, anti-oxidants, coloringagents, and sweeteners used regularly may be optionally added.

The intraoral strapping tablets are manufactured according to awell-known method. For example, they may be pharmaceuticallymanufactured as a mixture one or more active compound(s) with excipients(lactose, mannitol, glucose, microcrystal cellulose, colloidal silica,and starch, etc.), binders (hydroxypropylcellulose,polyvinylpyrrolidone, and magnesium metasilicate aluminate, etc.),disintegrator (starch, L-hydroxypropylcellulose, carboxymethylcellulose,crosscarmellose sodium, and cellulose calcium glycolate, etc.),lubricants (magnesium stearate etc.), swelling agents(hydroxypropylcellulose, hydroxypropylmethylcellulose, carbopole,carboxymethylcellulose, polyvinyl alcohol, xanthan gum, and CyamoposisGum, etc.), swelling adjuvants (glucose, fructose, mannitol, xylitol,erythritol, maltose, trehalose, phosphate salt, citrate, silicate,glycine, glutamate, and arginine, etc.), stabilizers, solubilizers(polyethylene glycol, propylene glycol, glutamate, and aspartic acid,etc.), or spices (orange, strawberry, mint, lemon, and vanilla, etc.),etc. according to usual methods. Further, they may be optionally coatedby coatings (sucrose, gelatin, hydroxypropylcellulose, andhydroxypropylmethylcellulose phthalate, etc.) or coated in the layer oftwo or more. Additives such as preservatives, anti-oxidants, coloringagents, and sweeteners used regularly may be optionally added.

The intraoral speed disintegrators are manufactured according to awell-known method. For example, they may be pharmaceuticallymanufactured as one or more active compound(s) itself/themselves or amixture bulk or granulated bulk particle with suitable coatings (ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, andacrylic acid methacrylate copolymer, etc.), plasticizers (polyethyleneglycol and triethyl citrate, etc.), excipients (lactose, mannitol,glucose, crystallite cellulose, colloidal silica, and starch, etc.),binders (hydroxypropylcellulose, polyvinyl pyrrolidone, and magnesiummetasilicate aluminate, etc.), disintegrators (starch,L-hydroxypropylcellulose, carboxymethylcellulose, crosscarmellosesodium, and cellulose calcium glycolate, etc.), lubricants (magnesiumstearate etc.), dispersant adjuvants (glucose, fructose, mannitol,xylitol, erythritol, maltose, trehalose, phosphate salt, citrate,silicate, glycine, glutamate, and arginine, etc.), stabilizers,solubilizer (polyethylene glycol, propylene glycol, glutamate, andaspartic acid, etc.), spices (orange, Strawberry, Mint, lemon, andvanilla, etc.) etc. according to usual methods. Further, they may beoptionally coated by coatings (sucrose, gelatin, hydroxypropylcellulose,and hydroxypropylmethylcellulose phthalate, etc.) or coated in the layerof two or more. Additives such as preservatives, anti-oxidants, coloringagents, and sweeteners used regularly may be optionally added.

The liquid compositions for oral administration include pharmaceuticallyacceptable waters, suspensions, emulsions, syrups, and elixirs, etc. Asto such liquid medicines, one or more active compound(s) may bedissolved, suspended, or emulsified to generally used diluent (purifiedwater, ethanol or those mixing liquids, etc.). Further, those liquidmedicines may contain humectants, suspending agents, emulsifying agents,sweeteners, flavor agents, flavoring agents, preservatives, and buffers,etc.

The external preparations for parenteral administration include, forexample, ointments, gels, creams, fomentations, patchs, embrocations,aerosols, inhalants, sprays, aerosols, eye drops, and nasal drops, etc.These include one or more activator(s) and are manufactured by awell-known method or the formula usually used.

The ointments include one or more activator(s) and are manufactured by awell-known method or the formula usually used. For example, they aremanufactured by levigating and melting one or more activator(s) tobasis.

The ointment bases are chosen from a well-known or the one usually used.They are used mixing one or two or more kinds chosen from, for example,higher fatty acid or higher fatty acid ester (adipic acid, myristicacid, palmitic acid, stearic acid, oleic acid, adipic acid ester,myristic acid ester, palmitate, stearic acid ester, and oleic acidester, etc.), rows (yellow wax, spermaceti, and ceresin, etc.),surfactants (polyoxyethylene alkyl ether phosphate etc.), higheralcohols (cetanol, stearyl alcohol, and cetostearyl alcohol, etc.),silicone oils (dimethylpolysiloxane etc.), hydrocarbons (hydrophilicpetrolatum, white petrolatum, purified lanolin, and liquid paraffin,etc.), glycols (ethylene glycol, diethylene glycol, propylene glycol,polyethylene glycol, and macrogol, etc.), vegetable oils (castor oil,olive oil, sesame oil, and oil of turpentine, etc.), animal oils (minkoil, yolk oil, squalane, and squalene, etc.), water, absorptionenhancer, and poisoned inhibitor. Further, they may include moisturizingagents, preservatives, stabilizing agents, anti-oxidants, and flavors,etc.

The gels are manufactured by a well-known method or a formula usuallyused. For example, they are manufactured by levigating and melting oneor more activator(s) to a basis. The base is chosen from a well-known orthe one usually used. It is used mixing one or two or more kinds chosenfrom, for example, lower alcohols (ethanol and isopropyl alcohol, etc.),gelatinizers (carboxymethylcellulose, hydroxyethyl cellulose,hydroxypropylcellulose, and ethyl cellulose, etc.), neutralizers(triethanolamine and diisopropanolamine, etc.), surfactants(mono-stearic acid polyethylene glycol, etc.), gums, water, absorptionenhancers, and poisoned inhibitors. Further, they may includepreservatives, anti-oxidants, and flavors, etc.

The creams are manufactured by a well-known method or a formula usuallyused. For example, they are manufactured by levigating one or moreactivator(s) to a basis and spreading and rolling on the support afterkneading. The base is chosen from a well-known or the one usually used.It is used mixing one or two or more kinds chosen from, for example,higher fatty acid esters, lower alcohols, hydrocarbons, polyhydricalcohols (propylene glycol, 1,3-butylene glycol, etc.), higher alcohols(2-hexyl decanol and cetanol, etc.), and emulsifiers (polyoxyethylenealkyl ethers and fatty acid esters, etc.), water, absorption enhancers,and poisoned inhibitors. Further, they may include preservatives,anti-oxidants, and flavors, etc.

The fomentations are manufactured by a well-known method or a formulausually used. For example, they are manufactured by levigating one ormore activator(s) to a basis and spreading and rolling on the supportafter kneading. The base is chosen from a well-known or the one usuallyused. It is used mixing one or two or more kinds chosen from, forexample, thickeners (polyacrylic acid, polyvinyl pyrrolidone, arabicgum, starch, gelatin, and methyl cellulose, etc.), humectants (urea,glycerin, and propylene glycol, etc.), and fillers (china clay, flowerof zinc, talc, calcium, and magnesium, etc.), water, absorptionenhancers, and poisoned inhibitors. Further, they may includepreservatives, anti-oxidants, and flavors, etc.

The patchs are manufactured by a well-known method or a formula usuallyused. For example, they are manufactured by levigating one or moreactivator(s) to a basis and spreading and rolling on the support afterkneading. The base for the pacth is chosen from a well-known or the oneusually used. It is used mixing one or two or more kinds chosen from,for example, high molecular basis, oils, fats, higher fatty acids,tackifiers, and poisoned inhibitors. Further, they includepreservatives, anti-oxidants, and flavors, etc.

The liniments are manufactured by a well-known method or a formulausually used. For example, they are manufactured by dissolving,suspending, or emulsifying one or more activator(s) to one or two ormore kinds chosen from water, alcohols (ethanol and polyethylene glycol,etc.), higher fatty acids, glycerins, soaps, emulsifiers, suspendingagents, etc. Further, they may include preservatives, anti-oxidants, andflavors, etc.

The aerosols, the inhalants, and the sprays may contain stabilizers suchas sodium hydrogen sulfite besides the diluent generally used, buffersgiving isotonicity, and isotonic agents such as, for example, sodiumchloride, sodium citrate, and citrates.

The injections for parenteral administration include solid shots thatare dissolved or suspended to solution, suspension, emulsion, or time ofuse solvent. The injections are used by dissolving, levigating andmelting one or more activator(s) to the solvent. As the solvent, forexample, water for injection, distilled saline, vegetable oil, propyleneglycol, polyethylene glycol, alcohols such as ethanol, etc., and thesecombination are used. Further, this injection may include stabilizers,solubilizers (glutamate, aspartic acid, and polysorbate 80 (registeredtrademark), etc.), suspending agents, emulsifying agents, soothingagents, buffers, and preservatives, etc. These are sterilize in thefinal process or are manufactured from the aseptic manipulation. Asepticsolid medicines may be manufactured as a freeze-drying product and maybe used by making to be aseptic or dissolving to aseptic distilled waterfor injection or other solvents before use.

The inhalant for parenteral administration includes aerosols, inhalantpowders, or inhalant solutions, which may be used by making to dissolveor suspend to water or other suitable media at the time of use.

These inhalants are manufactured according to a well-known method.

For example, the inhalant solutions are prepared by property selectingfrom preservatives (benzalkonium chloride and paraben, etc.), coloringagent, buffers (sodium phosphate and sodium acetate, etc.), tonicityagents (sodium chloride and concentrated glycerin, etc.), thickeners(carboxyvinyl polymer, etc.), and absorption enhancers, etc.,optionally.

The inhalant powders are prepared by properly selecting from lubricants(stearic acid and the salt, etc.), binders (starch and dextrin, etc.),excipients (lactose and cellulose, etc.), coloring agents, preservatives(benzalkonium chloride and paraben, etc.), and absorption enhancers,etc., optionally.

When the inhalant solution is administered, a sprayer (atomizer andnebulizer) is usually used. When the inhalant powder is administered, aninhalation administering machine for powder is usually used.

Other compositions for parenteral administration contain one or moreactivator(s) and include suppository for intrarectal administrationwhich is prescribed according to a routine procedure and pessary, etc.for intravaginal administering.

The sprays may contain a stabilizer such as sodium hydrogen sulfitebesides the diluent generally used, a buffer giving isotonicity, and anisotonic medicine such as, for example, sodium chloride, sodium citrate,or citrates. Production methods of the sprays have been described in,for example, U.S. Pat. No. 2,868,691 specification and U.S. Pat. No.3,095,355 specification in detail.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 (A) shows a flow cytometry that presents PD-1 expression inH-2L^(d)-specific 2C CTL clone and PD-L1 expression of P815 (cell linederived from mastocytoma) stable transfectant clone, and FIG. 1(B) showsa cytotoxic activity of 2C CTL cell line to PD-L1-expressing P815 celllines and the effect on the cytotoxic activity of anti-PD-L1 antibody(anti-PD-L1 F(ab′)₂ IgG).

FIG. 2(A)-FIG. 2(B) show tumor growth and infiltrating ofPD-L1-expressing P815 cell lines transplanted into syngeneic mouse. FIG.2(A) shows the tumour volume (upper) of the transplantedPD-L1-expressing P815 tumours and the survival rate (under) after thetransplant, FIG. 2(B) shows the tissue staining view of the transplantedPD-L1-expressing P815 tumour mass in syngeneic DBA/2 mouse (a shows aview (×40) that presents invasion of tumor cells across the abdominalwall and the peritoneum, b shows the same view (×400), c shows the viewof invasion into spleen, and d shows the view of invasion into liver.).

FIG. 3(A)-FIG. 3(B) show in vivo effect of anti-PD-L1 antibody for thetumor growth of the transplanted PD-L1-expressing P815 cell lines insyngeneic mice. FIG. 3(A) shows in vivo effect of anti-PD-L1 antibodyfor IFN-γ production from the tumor-specific CD8+ T cells in mice, FIG.3(B) shows in vivo effect of anti-PD-L1 antibody for the tumour volume(upper) and the survival rate (under) of the transplantedPD-L1-expressing P815 tumors in mice (squares show control group (ratIgG administrated group) and circles show anti-PD-L1 antibody(anti-PD-L1 F(ab′)₂ IgG) administrated group in the figure.).

FIG. 4 shows proliferation suppression of PD-L1-expressing B16 melanomasin homo-deficient syngeneic mice (PD-1−/−).

FIG. 5(A)-FIG. 5(C) show in vivo effect of anti-PD-L1 antibody to tumorgrowth of transplanted myeloma cell lines in syngeneic mouse (BALB/c)and the involvement of PD-1. FIG. 5(A) shows a flow cytometry that showsPD-L1 expression in various myeloma cell lines, FIG. 5(B) shows in vivoeffect of anti-PD-L1 antibody for tumor growth of J558L tumorstransplanted in the above mouse, and FIG. 5(C) shows comparison of tumorgrowth of J558L tumors transplanted in wild type and PD-1 gene deficient(PD-1−/−) syngeneic mice.

FIG. 6(A)-FIG. 6(B) show PD-L1 expression in capillary endothelium. FIG.6(A) shows PD-L1 and PD-L2 expression in vascular endothelial cells ofmice heart. FIG. 6(B) shows tissue staining of PD-L1 expression in eachtissue of mice. Each PD-L1 expression in (a): eyeball, (b):submandibular gland, (c): heart, (d): lungs, (e): liver, (f): kidney isshown. In the figure, Ch means choroidea, CV means middle cardiac vein,Gl means glomerule, and Re means retina. Each arrow shows vascularendothelial cells. Each staining image is enlargement view (×40).

FIG. 7(A)-FIG. 7(B) show PD-L1 expression in liver non-parenchymalcells.

FIG. 8 shows cell surface molecule phenotype in Kupffer cells and liversinusoidal endothelial cells (LSECs).

FIG. 9 shows cell surface molecule phenotype in CD4 positive T cells ofPD-1 gene homo-deficient mice (PD-1−/−) or wild type mice (wt).

FIG. 10 shows cell surface molecule phenotype in CD8 positive T cells ofPD-1 gene homo-deficient mice (PD-1−/−) or wild type mice (wt).

FIG. 11(A)-FIG. 11(B) show the effect of PD-L1 on T-cell proliferationin LNPC. FIG. 11(A) shows cellular proliferation when stimulating eachnaive T cells of PD-1−/− mice and wt mice. FIG. 11(B) shows the effectof anti-PD-L1 antibody on cellular proliferation in the co-culture of Tcells that has already been activated and derived from PD-1−/− mice orwt mice and LNPC.

FIG. 12(A)-FIG. 12(C) show the effect of PD-L1 on cytokine production inLNPC. FIG. 12(A) shows cytokine production when stimulating each naive Tcell of PD-1−/− mice and wt mice. FIG. 12(B) shows the effect ofanti-PD-L1 antibody on cytokine production in the co-culture of T cellsthat has already been activated and derived from PD-1−/− mice or wt miceand LNPC. FIG. 12(C) shows cell division of the activated T cells fromPD-1−/− mice or wt mice in the co-culture with LNPC.

FIG. 13(A)-FIG. 13(B) show the involvement of PD-1 in T lymphocytesproliferation in virus infected mouse liver. Cellular proliferation ofCD19- and CD3-positive lymphoid cells in liver and spleen of PD-1−/−mice or wt mice on FIG. 13(A) day 0 and FIG. 13(B) day 7 afteradenovirus infection are shown.

FIG. 14(A)-FIG. 14(B) show the involvement of PD-1 to cellularproliferation of T lymphocytes in virus infected mouse liver. FIG. 14(A)shows cellular proliferation of CD4- and CD8-positive lymphoid cells inliver of PD-1−/− mice or wt mice on day 7 after adenovirus infection.FIG. 14(B) shows the ratio of various proliferative lymphocytes on day 7after the infection.

FIG. 15 shows the involvement of PD-1 in virus infection. In thisfigure, (a)-(d) show tissue staining images that presents each cellularproliferation in liver of PD-1−/− mice and wt mice on day 0 and day 7after adenovirus infection. (e) and (f) show cellular proliferation ofCD4- and CD8-positive T cells in PD-1−/− mice on day 7 after adenovirusinfection.

FIG. 16 shows the involvement of PD-1 to virus infection. In thisfigure, (g)-(n) show hematoxylin & eosin tissue staining images in liverof PD-1−/− mice and wt mice on day 0 and day 7 after adenovirusinfection. (o)-(r) show X-gal tissue staining images in liver of PD-1−/−mice and wt mice on day 0 and day 7 after adenovirus infection.

FIG. 17(A)-FIG. 17(D) show the enhancement of each material to cytotoxicactivity. In this figure, FIG. 17(A) the enhancement of anti-mouse PD-1antibody, FIG. 17(B) the enhancement of anti-mouse PD-L1 antibody, FIG.17(C) the enhancement of mouse PD-1 Fc, and FIG. 17(D) the enhancementof human PD-1 Fc are shown.

BEST MODE FOR CARRYING OUT INVENTION

The following example explains the present invention more concretely,but do not limit the range of the present invention.

Example 1

A mouse PD-L1 expression vector was constructed by being inserted andligated mouse PD-L1 cDNA (Journal of Experimental Medicine (2000), vol.19, issue 7, p. 1027-1034.) digested with restriction enzyme EcoRI topApuroXS expression vector (The EMBO Journal (1994), vol. 13, issue 6,p. 1341-1349.). The expression vector pApuroXS-PD-L1 was transfectedinto P815 cells by electroporation (360V, 500 μF). The P815 cells werecultured in RPMI-1640 medium including FCS (10%), 2-mercaptoethanol(10⁻⁵M), and various antibiotics. The P815 cell lines which stablyexpressed mouse PD-L1 can be cloned by subculturing as resistant cellsunder medium including antibiotic puromycin (3 μg/ml). PD-L1 expressioncan be confirmed by flow cytometric analysis. FIG. 1(A) shows flowcytometries presenting (i) PD-1 expression of H-2L^(d)-specific 2C CTLclones and (ii) PD-L1 expression in PD-L1 expressing stable transformantof P815 (cell line derived from mastocytoma). Transformed B16 cell lines(B16/PD-L1) stably expressing PD-L1 were cloned by a similar method(FIG. 1(A) (iii)-(v)). pEFBOSneo-PD-L1 (Nucleic Acid Research (1990),vol. 18, issue 17, p. 5322.) constructed as a expression vector by asimilar method was used, and G418 (0.5 mg/ml) was used for selectiveculture of the cell lines.

cDNA coding protein to connect tandem 6×his peptide tag (His-Tag) to 3′end side of total length mouse PD-L1 cDNA was inserted in expressionvector pVL 1393 (the trade name: purchase from Clonetech) after beingdigested by restriction enzyme EcoRI and NotI. Then, this expressionvector was continuously introduced to SF9 insect cells (purchased fromInvitrogen), and the inclusion body was collected. This inclusion bodyvirus was infected to HiFive insect cells (purchased from Invitrogen) byculturing for 2 days at 27° C. The purified PD-L1 protein that are usedas antigen was obtained by processing cell lysate dissolved with buffersolution (Tris-HCl (50 mM, pH7, and 1% TritonX-100), EDTA (10 mM), andNaCl (150 mM), various protease inhibitors) with Ni-sepharose columnchromatography.

The dialyzed PD-L1 protein was immunized to female wister rat at 8 weeksage (purchased from SLC Japan) with a complete Freund adjuvant(purchased from Amersham) and after several days, 2×10 of cellscollected from peripheral lymph node were fused with the same number ofSP2/0 cells using PEG1500. Further, the fused cells were selected byculturing in RPMI1640 medium (HAT (purchased from Sigma), Origen (10%,purchased from Igen), and FCS (10%), 2-mercaptoethanol (10⁻⁵M), andvarious antibiotics), and the presence of the antibody production wasconfirmed by flow cytometric analysis. The monoclonal antibody (1-111)to PD-L1 was obtained by purification of the collected peritoneal fluidwith protein G sepharose column chromatography after transfering theestablished hybridoma (hybridomas recognized with International TrustNumber: FERM BP-8396) to Balb/C nu/nu mice. As antibodies used by flowcytometry, etc., biotinated antibodies using Sulfo-NHS-LC-biotin (thetrade name: purchased from Pierce) can be used.

Further, anti-human PD-1 antibody (the monoclonal antibody productedfrom hybridomas recognized with International Trust Number: FERMBP-8392) was prepared according to a similar method.

Cytotoxicity assay was executed by ⁵¹Cr (chromium) separation assay.

2C cells (Journal of Immunology (1996), vol. 157, issue 2, p. 670-678.)are (H-2L)^(d) alloreactive cytotoxic T-cells derived from 2C transgenicB6 mice. After mixing 2C cells (E: effector) together with ⁵¹Cr-labeledP815 cells (T: target) (circle), three kinds of PD-L1-expressing P815cells (P815/PD-L1) (square, diamond, and triangle) respectively, orfurther under the presence of 10 mg/ml rat anti-PD-L1 F(ab′)₂ IgG(filled triangle), a result of measuring separated ⁵¹Cr for 4 hours withvarious E/T ratio is shown in FIG. 1 (B).

Anti-PD-L1 antibody (anti-PD-L1 F(ab′)₂) recovered the decreasedcytotoxic activity of cytotoxic T lymphocytes. Those results suggestedthat inhibition of PD-1 and PD-L1 signal by inhibiting PD-L1 functioncould reinforce cytotoxic activity to carcinoma cells.

Example 2

The tumor growth and the survival rate of mice was evaluated byhypodermically transfering 1×10⁶ of P815 cells (n=6) or P815/PD-L1 cells(n=6) to syngeneic DBA/2 mice respectively. The result is shown in FIG.2 (A). In this figure, circles show P815 cell lines transplanted group,and squares and triangles show PD-L1-expressing P815 cell linestransplanted group respectively. Further, histological analysis ofP815/PD-L1 cells transferred group was executed. FIG. 2(B) showsstaining images dyed with hematoxylin & eosin after being fixed by 10%formaldehyde and embedded into paraffin. In this figure, a shows a view(×40) that presents invasion of tumor cells across the abdominal walland the peritoneum, b shows the same view (×400), c shows the view ofinvasion into spleen, and d shows the view of invasion into liver.

In the P815 cells transplanted group, P815 cells proliferation wassuppressed and 30 percent of mice in this group lived by 6-7 weeks,while in PD-L1-expressing P815 cells (P815/PD-L1) transplanted group,carcinoma cells proliferation was remarkable and all mice died by 2-4weeks (FIG. 2(A)). P815/PD-L1 cells were observed to permeate across theperitoneal cavity and the abdominal cavity, and further those metastasesto liver and spleen (FIG. 2(B) a-d).

Example 3

After co-culturing 2×10⁶ of 2C cells together with 5×10⁶ of P815 cellsor P815/PD-L1 cells only, or P815/PD-L1 cells under the presence of 10mg/ml rat anti-PD-L1 F(ab′)₂ IgG respectively, IFN-γ in culturesupernatant at 24 hours was measured with ELISA kit (purchased fromBioscience). The result is shown in FIG. 3 (A).

FIG. 3 (B) shows the result of evaluating of the tumor growth and thesurvival rate of mice of which anti-rat IgG (square) or anti-PD-L1F(ab′)₂ IgG (0.1 mg/mouse) (circle) was intraperitoneally administeredto syngeneic DBA/2 mice (n=10) to which 3×10⁶ of P815/PD-L1 cells hadbeen hypodermically transferred on day 1, 3, 5, and 7 after thecell-transfer.

Anti-PD-L1 antibody restored IFN-γ production from cytotoxic Tlymphocytes which had been suppressed by P815/PD-L1 (FIG. 3(A)).Administration of anti-PD-L1 antibody suppressed carcinoma cell growth,and showed the clear survival effect (FIG. 3(B)). This result presentsthat administration of anti-PD-L1 antibody is effective on cancertreatment.

Example 4

1×10 of B16 melanomas (n=6) or B16/PD-L1 cells (n=6) were hypodermicallytransferred to B6 mice (n=6) respectively, and the same number ofB16/PD-L1 cells were transferred to PD-1 transgenic B6 mice (n=5) andPD-1 gene homo-deficient B6 mice (PD-1/−(n=4)) (Science(2001), vol. 291,issue 5502, p. 319-332.), each tumor growth was measured by 25 daysthereafter. FIG. 4 shows the result.

Example 5

Tumor growth of which (n=9) anti-rat IgG or anti-PD-L1 F(ab′)₂ IgG (0.1mg/mouse) was intraperitoneally administered to syngeneic Balb/C mice towhich 2.5×10⁸ of J558 myeloma cells had been hypodermically transferredon day 3, 5, and 7 after the cell-transfer was evaluated. Each tumorgrowth in PD-1 gene homo-deficient Balb/C mice and Balb/C mice (n=4) towhich J558 myeloma cells had been hypodermically transferred werecompared (FIG. 5(B)).

Administration of anti-PD-L1 antibody suppressed PD-L1-expressing J558carcinoma cells proliferation (flow cytometries of PD-L1 expression invarious myeloma cell lines are shown in FIG. 5(A))(FIG. 5(B)). Thetransplanted tumor cells proliferation was completely inhibited inPD-1-deficient mice to which J558 cells had been transplanted (FIG.5(c)). These results present that inhibition of PD-L1 or PD-1 iseffective on cancer treatment.

Example 6

Vascular endothelial cells (hereafter, abbreviated as ECs) was gatheredfrom mouse heart by the method of Marelli-Berg (Journal of immunologicalmethods (2000), vol. 244, issue 1-2, p. 205-215.). Concretely, hearttissue digested by collagenase was pre-cultured followed by beingpre-cultured with mouse Ig and co-cultured together with FITC-modifiedanti-CD3 antibody, the same modified anti-CD105 antibody, the samemodified anti-isolectin B4 antibody, and anti-FITC bead. These vascularendothelial cells were purified by positive selection usingMagnetic-activated cell-sorting separation columns (the trade name:purchased from Miltenyi Biotec).

PD-L1 and PD-L2 expressions in the gathered vascular endothelial cellswere confirmed by flow cytometry. The cell labelling was executed usinganti-PD-L1 antibody (the antibody name: 1-111), anti-PD-L2 antibody (theantibody name: #122), and fluorescent labelling second antibody (FIG.6(A)). The analysis was executed 10,000 events with Facscalibur (theequipment name: purchased from Becton Dickinson) using CellQuestsoftware (purchased from Dickinson). PD-L1 or PD-L2 expression is shownas shedding curve, and control Ig is shown as filling curve.

PD-L1 expression in each mouse tissue was confirmed by tissue staining.Before 1 hour each tissue sampling, 100 μl of PBS which 100 μg ofbiotin-labelling anti-PD-L1 antibody (1-111) had been dissolved wasintravenously administered to mouse. Then, 5 μm frozen section was fixedby 4% paraformaldehyde (PFA), and dyed with Streptavidin-FITC. Eachsection was countered staining by Phalloidin (FIG. 6(B) shows PD-L1expression in (a) eyeball, (b) submaxillary gland, (c) heart, (d) lung,(e) liver, (f) kidney. In this figure, Ch presents choroid, CV presentsvena centralis, Gl presents glomerule, and Re presents retina. Eacharrow presents vascular endothelial cells. Each staining figure is 40×enlargement view.). PD-L1 expression was observed in capillaryendothelium of heart, lung, kidney, stomach, small intestine,submandibular gland, eyeball, and liver. The expression in liver waslocalized in liver sinusoidal capillary.

Example 7

PD-L1 expression in liver non-parenchymal cells (hereafter, abbreviatedwith LNPCs) was confirmed by tissue staining (FIG. 7(A)) and flowcytometry (FIG. 7(B)). In the tissue staining, 5 μm liver frozen sectionfixed with 3% of PFA was preprocessed by rat serum followed by beingreacted for 1 hour at room temperature using biotin-labeled anti-PD-L1antibody (1-111) or biotin-labeled anti-ICAM-1 antibody (the trade name:purchased from BD Pharmingen), and then the biotin antibodies werevisualized by tyramide signal amplification (TSA) fluorescence system(the equipment name: purchased from PerkinElmer Life Sciences) (FIG.7(A) shows ICAM-1 expression, FIG. 7(B) shows PD-L1 expression, and CVpresents vena centralis. Each staining figure is 40× enlargement view.).

LNPCs were isolated from mouse liver according to the pronaseE method(Experimental Cell Research (1976, vol. 99, p. 444-449.). Concretely,LNPCs obtained by which liver is circulated with pronaseE solution(Merck) were cultured and were separated by density gradientcentrifugation. Relative distribution of Kupffer cells (CD54+, CD11bhigh) in the cell suspension is 20-25%, and that of liver perisinusoidalspace endothelial cells (hereafter, abbreviated with LSECs.) (CD54+,CD11b high) is 75-80%. Kupffer cells and LSECs were doubly stained usingFITC-labeled anti-CD11b antibody, each biotinated monoclonal antibody toICAM-1, PD-L1, B7-1, and B7-2, and PE-labeled Streptavidin,respectively. Kupffer cells and LSECs were gate as CD11b high and CD11blow cell, respectively (FIG. 8).

PD-L1 expressed together with ICAM-1, B7-1, and B7-2 in Kupffer cells,while the expression was weak in LSECs (FIG. 8).

Example 8

Native T cells were refined (refining degree 90% or more) from spleenand lymphoid of PD-1 gene homo-deficient mice (PD-1−/−) or wild typeC57BL/6 mice (wt) by a negative selection using T-cell enrichment column(the trade name: purchased from Genzyme). The cells cultured for 48hours with 10 μg/ml of anti-CD3 monocional antibody (2C11) wereactivated. The naive T cells activated by the above method were doublystained using FITC-labeled anti-CD4 antibody or APC-labeled anti-CD8antibody, PE-labeled anti-CD25 antibody, PE-labeled anti-CD4 antibody,PE-labeled anti-CD69 antibody, or PE-labeled anti-CTLA-4 antibody,biotin-labeled anti-B7-1 (CD80) antibody or biotin-labeled anti-B7-2(CD86) antibody, and anti-PD-1 antibody (the antibody name: J43,monoclonal antibody producted from hybridomas recognized byInternational Trust Number FERM BP-8118) or anti-PD-L1 antibody (1-111),and each molecule expressions were analyzed by flow cytometry (FIGS. 9and 10).

The hybridomas identified by International Trust Number FERM BP-8118 hadbeen deposited as Trust Number FERM P-18356 to National Institute ofAdvanced Industrial Science and Technology, International PatentOrganism Depositary in Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan(ZIP code 305-8566) at May 30, 2001, and was transferred toInternational Deposit at Jul. 16, 2002.

Example 9

The naive T cells activation of PD-1 gene homo-deficient mice (PD-1−/−)or wild type mice (wt) was executed by the method described in example8. Proliferation of these activated cells was measured by BrdUincorporation method (FIG. 11(A)). The proliferation was measured byadding BrdU at the last 6 hours of 48 hours to label the cells andmeasuring using Proliferation ELISA kit (the trade name: Purchased fromRoche). The amount of IFN-γ production at this time was measured inELISA Kit (the trade name: purchased from Genzyme) (FIG. 12(A)).

T cells derived from PD-1 gene homo-deficient mice (PD-1−/−) or wildtype mice (wt) was beforehand activated by the method described inexample 8. The activated T cells were respectively cultured for 60 hoursunder the presence or the absence of Mitomycin C-processed LNPCs of wildtype mice and under the presence or the absence of 30 μg/ml ofanti-PD-L1 antibody (1-111) (rat IgG as control) and 20 μg/ml ofCTLA4-Ig (Genzyme) (human IgG as control), and the cells proliferationfor the last 12 hours was measured by BrdU incorporation method (FIG.11(B)). Further, the amount of IFN-γ production at 48 hours was measured(FIG. 12(B)).

As to the amount of IFN-γ production when naive T cells were activated,a significant difference between PD-1−/− and wild type mice was notobserved. On the other hand, in the activated T cells, the amount ofIFN-γ production of T cells derived from wild type mice wassignificantly lower than that derived from PD-1−/− (FIG. 12). Therefore,it was suggested that the effect of PD-1 inhibitory action on theactivated T cells could be higher than the effect on naive T cellsactivation.

In co-culture of the activated T cells derived from wild type mice andLNPCs, a significant difference in the cellular proliferation and theamount of IFN-γ production of these T cells was not observed, while inthe co-culture of the activated T cells derived from PD-1−/− mice andLNPCs, a significant increase in the cellular proliferation of these Tcells was observed (FIG. 11(B). FIG. 12(B)). In adding anti-PD-L1antibody to the co-culture of the activated T cells derived from wildtype mice and LNPCs, an increase in the cellular proliferation of theseT cells was observed (FIG. 11(B)). These results suggest that PD-1 orPD-L1 of LNPCs could take part in suppression of T cells activation andlack of PD-1 or inhibition of interaction between PD-1 and PD-L1 couldactivate T cells.

The activated T cells of PD-1 gene homo-deficient mice (PD-1−/−) or wildtype mice (wt) was labeled by 5 μM CFSE (5-(6)-carboxy-fluoresceindiacetate succinimidyl diester) (the trade name: purchased fromMolecular probes) and was co-cultured together with LNPCs for 48 hours.Cell division at this time was decided by CFSE activity measurementusing FACS (FIG. 12(C)).

It was suggested that the cellular proliferation suppression of theactivation T cells could cause in cytostasis suppression and PD-1 signalcould suppress cell division of T cells (FIG. 12(C)).

Example 10

PD-1 gene homo-deficient mice (PD-1−/−) or wild type mice (wt) (3 per agroup) were infected in adenoviruses by intravenous administration of10⁹-10¹⁰ PFU (plaque-forming units) of Ad-lacZ. Ad-lacZ used here, whichare 5 type adenovirus with lacZ gene, lacks E1 and E 3 region and can bepurified by cesium chloride density-gradient centrifugation (NucleicAcid Research (1995), vol. 234, issue 19, p. 3816-3821.) afterproliferating them in 293 cells. On day 0 or 7 after infection, spleencells and intrahepatic lymphocytes that had been gathered afterintravenous administration of 0.5 mg of BrdU (the trade name: purchasedfrom Sigma) to the mice at 1 hour before slaughtering were doublylabeled by anti-BrdU antibody and anti-CD19 antibody, or anti-CD3antibody (FIG. 13).

The cells on day 7 after infection were doubly labeled by anti-BrdUantibody, anti-CD19 antibody, anti-CD3 antibody, anti-CD4 antibody, andanti-CD8 antibody (FIG. 14(B), each bar graph shows the ratio ofBrdU-positive cells.).

In adenovirus infected PD-1−/− mice liver, the ratio of eachproliferative (BrdU positive) lymphocyte (CD19 positive, CD3 positive,CD4 positive, or CD8 positive) had increased compared to that insimilarly infected wild type mice liver. On the other hand, since such aphenomenon in spleen was not observed, it was suggested that PD-1 couldinhibit T cells proliferation in inflammatory tissues (FIG. 14(B)).

Example 11

On day 0 or 7 after 10⁹-10¹⁰ PFU of Ad-lacZ had been intravenouslyadministered to PD-1 gene homo-deficient mice (PD-1−/−) or wild typemice (wt) (3 per a group), liver slices that had been gathered afterintravenous administration of 0.5 mg of BrdU (the trade name: purchasedfrom Sigma) to the mice at 1 hour before slaughtering were doublylabeled by anti-BrdU antibody (FIG. 15(a)-(d), 20× enlargement view.).Liver slices of PD-1 gene homo-deficient mice (PD-1−/−) on day 7 afterinfection were doubly labeled by anti-BrdU antibody, anti-CD19 antibody,anti-CD3 antibody, anti-CD4 antibody, and anti-CD8 antibody (FIG. 15(e),(f), 40× enlargement view.).

In wild type mice liver on day 30 after infection, the locally andmedium cell invasion to sinusoidal capillary and non-parenchymal regionwas observed, but in PD-1−/− mice, hepatitis symptom was not observed(FIGS. 16(h), (i) and (i), (n)).

Liver slices of PD-1 gene homo-deficient mice (PD-1−/−) or wild typemice (wt) on day 7 or 30 after infection were stained by hematoxylin &eosin (FIG. 16(g)-(j), 20× enlargement view, (k)-(n), 40× enlargementview.) and X-gal (FIG. 16 (o)-(r), 40× enlargement view.). In liver ofwild type mouse on day 7 and 30 after infection, adenovirus infectionindicated by X-Gal was confirmed, while the infection in that ofPD-1^(−/−) mouse had been almost eliminated on day 30 (FIG. 16 (o), (p),(q), and (r)). These results presented that PD-1 signal could take partin the exclusion of viruses by which the proliferation of effector Tcells in virus infected tissue could be induced.

Example 12

P-815/PD-L1 cells compellingly expressing mouse PD-L1 were sown intoculture flask and were cultured in usual medium including 5 μg/mL ofpuromycin (purchased from Sigma)(hereafter, abbreviated as selectivemedium) at 37° C. under 5% CO₂/95% air until to be 50%-90% confluent.Mouse cytotoxic T lymphocyte 2C cells were subcultured for several daysin usual medium together with P-815 cells processed by MMC (Mitomycin C)and culture supematant of ConA-stimulated rat splenic cells. Thecollected P-815/PD-L1 cells were cultured for 15 minutes after adding 3μL of BATDA Reagent of DELFIA EuTDA Cytotoxicity Reagents (purchasedfrom PerkinElmer). Then, they were washed with PBS. 2C cells subculturedfor 5-8 days after adding P-815 cells were used.

As subject substrates, 20 μL (10 ng/mL) of anti-mouse PD-1 antibody(figure. 17, anti-mPD-1Ab(J43)), anti-mouse PD-L1 antibody(anti-mPD-L1Ab(1-111) in this figure), mouse PD-1 Fc (mPD-1Fc in thisfigure), human PD-1 Fc (hPD-1Fc in this figure), mouse IgG2aK (ControlIg in this figure), or PBS were dispensed into 96 well plate, and then50 μL of P-815/PD-L1 cells or usual medium were added. Further, 50 μL of2 C cells, usual medium, or usual medium including 1% Triton ×100 wereadded. 50 μL of supernatants in wells which the usual medium were addedwere collected for background and other supernatants were preserved at37° C. until being collected. The residual cells were cultured for 4hours. The 96 well plates were centrifuged and the supernatants werecollected. 200 μL of DELFIA Europium Solution of Cytotoxicity Reagents(purchased from PerkinElmer) were added to the collected supernatants,and were shaken for 15 minutes. After shaking, time resolvedfluorescence measurement was executed with ARVOsx multi-label counter(WALLAC). The supernatants in wells that the usual medium including 1%Triton ×100 was added were used as a high control, and the one in wellsthat the usual medium were used as a low control.

The evaluated group is composed of a subject substrate, P-815/PD-L1cells, and 2C cells, the high control group is composed of PBS,P-815/PD-L1 cells, and usual medium including 1% Triton ×100, and thelow control group is composed of PBS, P-815/PD-L1 cells, and usualmedium, the 2C cell control group is composed of PBS, usual medium, and2C cells, and the background group is composed of PBS, P-815/PD-L1cells, and usual medium. CTL activity (%) was calculated by thefollowing formula. All values are the one that had been subtracted bythe average of the background.

CTL activity (%)=([the measurement of the evaluation group]−[themeasurement of the 2C cell control group]−[the measurement of the lowcontrol group])/([the measurement of the high control group]−[themeasurement of the low control group])×100

Anti-PD-1 antibody, anti-PD-L1 antibody, and PD-1 Fc have significantlyreinforced CTL activity (FIG. 17 (a)-(d), E: T ratio presents the mixingratio of 2C cells and PD-L1/P815 cells.).

Example 13

The inhibiting effect of anti-PD-1 antibody on cancer metastasis wasevaluated by intraperitoneal administration of anti-mouse PD-1monoclonal antibody to C57BL/6 mice to which B16 melanoma cells had beentransferred at intervals of 2 days followed by measuring the liverweight on day 18 after transfer.

The increase in the liver weight of anti-PD-1 antibody administratedgroup was significantly suppressed than that in control IgGadministrated control group (liver weight/carcinoma cell non-transferredgroup: 1.3 g, decrease from control group: 6.8 g to anti-PD-1 antibodyadministrated group: 3.5 g.). The suppression of the increase in thisweight presents that the metastasis of B16 melanoma cells is suppressed.

1-32. (canceled)
 33. A method of treating a tumor in a human in needthereof comprising administering to the human an effective amount of ahuman or humanized anti-PD-L1 monoclonal antibody that inhibits aninteraction between PD-1 and PD-L1 in combination with an effectiveamount of an anti-CTLA-4 antibody.
 34. The method of claim 33, whereinthe anti-PD-L1 monoclonal antibody comprises a heavy chain constantregion of an IgG1, IgG3, or IgG4 isotype.
 35. The method of claim 33,wherein the anti-PD-L1 monoclonal antibody comprises a heavy chainconstant region of an IgG1 isotype.
 36. The method of claim 33, whereinthe tumor is a solid tumor.
 37. The method of claim 33, wherein theanti-PD-L1 monoclonal antibody suppresses growth of the tumor ormetastasis of the tumor.
 38. The method of claim 33, wherein the tumoris one or more selected from a carcinoma, a squamous carcinoma, anadenocarcinoma, a sarcoma, a leukemia, a neuroma, a melanoma, and alymphoma.
 39. The method of claim 38, wherein the squamous carcinoma isin the cervical canal, eyelid, tunica conjunctiva, vagina, lung, oralcavity, skin, urinary bladder, tongue, larynx, or gullet.
 40. The methodof claim 38, wherein the adenocarcinoma is in the prostate, smallintestine, endometrium, cervical canal, large intestine, lung, pancreas,gullet, intestinum rectum, uterus, stomach, mammary gland, or ovary. 41.The method of claim 33, wherein the tumor is melanoma.
 42. The method ofclaim 33, wherein the tumor is in the lung.
 43. The method of claim 33,wherein the tumor is in the bladder.
 44. The method of claim 33, whereinthe tumor expresses PD-L1.
 45. The method of claim 33, furthercomprising administering a chemotherapeutic drug.
 46. The method ofclaim 33, wherein the anti-CTLA-4 antibody is administered to the humantogether with the anti-PD-L1 monoclonal antibody.
 47. The method ofclaim 33, wherein the anti-CTLA-4 antibody is administered to the humantogether as a mixture with the anti-PD-L1 monoclonal antibody.
 48. Themethod of claim 42, wherein the anti-CTLA-4 antibody is administered tothe human together with the anti-PD-L1 monoclonal antibody.
 49. A methodof suppressing tumor growth in a human in need thereof comprisingadministering to the human an effective amount of a human anti-PD-L1monoclonal antibody in combination with an effective amount of ananti-CTLA-4 antibody, wherein the anti-PD-L1 monoclonal antibody and theanti-CTLA-4 antibody suppress the tumor growth in the human.
 50. Themethod of claim 49, wherein the anti-PD-L1 monoclonal antibody inhibitsan interaction between PD-1 and PD-L1 in suppressing the tumor growth.51. The method of claim 49, wherein the tumor is a solid tumor.
 52. Themethod of claim 51, wherein tumor metastasis is suppressed.
 53. Themethod of claim 49, wherein the tumor is one or more selected from acarcinoma, a squamous carcinoma, an adenocarcinoma, a sarcoma, aleukemia, a neuroma, a melanoma, and a lymphoma.
 54. The method of claim53, wherein the squamous carcinoma is in the cervical canal, eyelid,tunica conjunctiva, vagina, lung, oral cavity, skin, urinary bladder,tongue, larynx, or gullet.
 55. The method of claim 53, wherein thesquamous carcinoma is in the lung.
 56. The method of claim 53, whereinthe adenocarcinoma is in the prostate, small intestine, endometrium,cervical canal, large intestine, lung, pancreas, gullet, intestinumrectum, uterus, stomach, mammary gland, or ovary.
 57. The method ofclaim 49, wherein the tumor is melanoma.
 58. The method of claim 49,wherein the tumor is in the lung.
 59. The method of claim 49, whereinthe tumor is in the bladder.
 60. The method of claim 49, wherein thetumor expresses PD-L1.
 61. The method of claim 49, wherein theanti-CTLA-4 antibody is administered to the human together with theanti-PD-L1 monoclonal antibody.
 62. A method of treating a lung cancerin a human in need thereof comprising administering to the human aneffective amount of a human anti-PD-L1 monoclonal antibody of an IgG1isotype that inhibits an interaction between PD-1 and PD-L incombination of an effective amount of an anti-CTLA-4 antibody, whereinthe administering treats the lung cancer.